The molecular origins of sickle cell disease ("SCD") are rooted in the tetrameric, .alpha..sub.2.beta..sub.2, hemoglobin molecule, the blood's O.sub.2 and CO.sub.2 molecular transporter. Pauling et al, 110 Science 543-48 (1949). SCD is believed to be caused by a single point mutation in the gene coding for the .beta. polypeptide chain of hemoglobin A ("HbA"), resulting in an amino acid change from glutamic acid to valine at position 6. Individuals who are homozygous for the hemoglobin S ("HbS") allele suffer severe medical consequences. In contrast, individuals who are heterozygous are rarely affected. The medical consequences arise because the deoxygenated form of HbS ("deoxy-HbS") exhibits a dramatic reduction in solubility relative to the deoxygenated form of HbA ("deoxy-HbA"). Deoxy-HbS polymerizes into aggregating fibers, which form a viscous gel that distorts the normally biconcave disc shape of normal erythrocytes into a characteristic banana or "sickle" shape. Wellems et al., 135 J. Mol. Biol. 651-74 (1979). The sickled cells are more rigid and less deformable than normal erythrocytes, which causes hemolysis and microvascular occlusion and leads to numerous pathologies including painful crises, organ damage and a reduced life expectancy. Dean et al., 299 New Engl. J. Med. 752-63, 804-11, 863-70 (1978); Hebbel, 77 Blood 214-37 (1991).
The rate of deoxy HbS polymerization is highly dependent on the initial cellular concentration of HbS, such that small decreases in the concentration of HbS tetramers capable of polymerizing can dramatically slow the rate of deoxy-HbS polymerization. Hofrichter et al., 73 Proc. Nat. Acad Sci., U.S.A. 3035-39 (1976); Eaton et al., 70 Blood 1245-66 (1987); Hofrichter et al., 71 Proc. Nat. Acad Sci. U.S.A. 4864-68 (1974). It has been suggested that a 15-20% decrease in the HbS population participating in polymer formation may decrease the severity of the disease to that of the less severe S/.beta..sup.+ -thalasemia, and be of significant therapeutic value. Sunshine et al., 275 Nature 238-40 (1978). Various chemotherapeutic approaches have been based on this strategy.
The problem of finding potent noncovalent gelation inhibitors for possible development in the treatment of sickle cell disease has been evident for over a quarter of a century. Gorecki et al., 77 Proc. Nat. Acad. Sci., U.S.A. 181-85 (1980). The molecular target, HbS, is well known and in abundant supply. Nonetheless, there are no anti-sickling agents to date approved for clinical use. There are at least two compounds undergoing serious clinical evaluation as therapeutics for SCD: a surfactant, poloxamer 188, for reducing the stickiness of sickle cells, and hydroxyurea for enhancing the production of fetal Hb, an effective diluent and inhibitor of the polymerizable HbS. Adams-Graves et al., 90 Blood 2041-46 (1997); Bridges et al., 88 Blood 4701-10 (1996); Charache et al., 332 New Engl. J. Med. 1317-22 (1995); Platt, 74 J. Clin. Invest. 652-56 (1984).
An alternative therapeutic strategy involves the use of small organic ligands that bind to HbS and interfere with the polymerization reaction. One starting point in a search for such molecules has been the use of high resolution crystal structures of small molecule-hemoglobin complexes. In that approach, compounds that hinder HbS polymerization were designed, yielding a number of compounds that have clear activity in inhibiting HbS polymerization. Abraham et al., 25 J. Med. Chem. 1015-17 (1982). However, with the exception of a number of ethacrynic acid analogues, most would have to be administered at rather high (ca. 5 mM) concentrations to achieve a therapeutically significant effect. Abraham et al., 32 J. Med. Chem. 2460-67 (1989); Abraham et al., 27 J. Med. Chem. 967-78 (1984); Abraham et al., 27 J. Med. Chem. 1549-59 (1984); Fatope et al., 30 J. Med. Chem. 1973-77 (1987).
Small organic molecules have been used to bind to HbS noncovalently, thereby inhibiting the intermolecular interactions required for aggregation. Gorecki et al., 77 Proc. Nat. Acad. Sci., U.S.A. 181-85 (1980); Abraham et al., 27 J. Med. Chem. 967-78 (1984); Belie et al., 18 Biochemistry 4196-201 (1979); Elbaum et al., 71 Proc. Nat. Acad. Sci., U.S.A. 4718-22 (1974); Noguchi et al., 17 Biochemistry 5455-59 (1978); Ross et al., 77 Biochem. Biophys. Res. Comm. 1217-23 (1977). However, most compounds examined to date have shown limited potency in enhancing deoxy-HbS solubility. This modest success is the result, in part, of limitations in screening. Existing functional assays, like the C.sub.sat assay to determine HbS solubility, Mazhani et al., Hemoglobin, 129-136 (1984), are too cumbersome to permit extensive screening.
The development of new small molecule therapeutics typically begins with the identification of an active, or lead, compound that exhibits some of the properties required for safe and effective therapeutic intervention. Compounds with improved properties are subsequently derived through iterative cycles of analog preparation and testing. Lead compounds are often identified using high throughput screening (HTS), whereby large libraries containing 50,000 to 1,000,000 compounds are tested using relatively simple assays to measure inhibition of processes critical to the target indication. Typically this means using biochemical assays to measure the function of one or more macromolecular targets.
However, functional assays appropriate for HTS can be difficult to establish as they require that: the biochemical process is well understood; the molecular participants are identified and available, often in large quantities; and the technologies needed to measure the relevant biochemical events are compatible with HTS. Such assays may also oversimplify the targeted process by probing only one function of a multifunctional macromolecular target. Sometimes assays based on intact cells can be used, allowing many aspects of a process to be targeted simultaneously. However, such measurements often generate less reproducible data than high quality biochemical assays and only detect active compounds that readily enter the cells and remain stable to cellular metabolic processes. Very often interesting compounds are identified in cell-based assays, but further development is impeded by an inability to identify the specific molecular target.
Many of the difficulties associated with HTS based on complex biochemical or cell-based assays can be circumvented by using an affinity-based primary screen. This is a universal approach since all lead compounds share the common property that they must bind to their macromolecular targets. Screening by affinity identifies a small subset of ligands which can be subsequently tested for function using a variety of low throughput biochemical and cell-based assays. Complex functional assays, unsuitable for high throughput use, can be employed to identify active compounds among the members of the smaller, more manageable ligand library. All ligands from the original library should be identified. Therefore, when multiple biochemical activities of the target are known, all the activities of each ligand can be measured separately without the prohibitive effort that would be needed to screen the entire library using multiple functional assays. When cell-based assays are available, affinity prescreening allows high throughput discovery even for macromolecular targets whose biochemical activities are uncharacterized. In contrast to the case for cell-based primary screens, determination of the molecular target of active compounds identified through cell-based secondary assays of known ligands is usually unnecessary.
A variety of affinity screening approaches have been described, including several strategies involving solid phase display of synthetic compounds produced through combinatorial synthesis, bacteriophage and bacterial display of random peptide libraries, and in vitro selection of RNA and DNA ligands to target proteins. Terrett et al., 51 Tetrahedron 8135-73 (1995); Scott et al., 249 Science 386-90 (1990); Lu et al., 13 Bio/Technology 366-72 (1995); Tuerk, 67 Meth. Mol. Biol. 219-30 (1997). Each of these methods significantly limits the types of compounds that can be assayed. None are compatible with screening natural product extracts or the vast collections of individually synthesized small molecules that are the traditional sources of compound diversity employed in drug discovery. Since these methods involve separation steps to differentiate bound from unbound material, they are unable to detect the binding of ligands whose dissociation half lives are significantly shorter than the time of separation. Ligands with micromolar dissociation constants typically dissociate with half lives far shorter than one second; therefore, only very high affinity ligands can usually be detected.
High throughput binding assays that require no separation steps have generally been unavailable. Recently, methods have been described wherein mass spectroscopy is used to directly measure protein-ligand complexes. Loo, 16 Mass Spec. Rev. 1-23 (1997). However, because there can be large variations in the stability of complexes upon desolvation from solution to the gas phase of the mass spectrometer, the generality of this approach is uncertain.
Accordingly, it can be seen that there is a need for new methods for selecting new small molecule thereapeutics for use in the treatment of sickle cell disease. Further, it can be seen that there is a need for new compounds useful in pharmaceutical formulations for treating sickle cell disease.